Identification of Pseudomonas strains for the biological control of soybean red crown root rot

Soybean red crown root rot (RCR), caused by the soil-borne fungal pathogen, Calonectria ilicicola, is the most destructive disease affecting soybean production in Japan. To date, no resistant cultivars or effective fungicides have been developed to control this disease. In this study, we evaluated 13 bacterial strains to determine their efficacy in controlling C. ilicicola. We first investigated whether the volatile organic compounds (VOCs) emitted by the bacterial strains exhibited any antifungal activity against C. ilicicola using the double-plate chamber method. The results showed that VOCs from three Pseudomonas bacterial strains, OFT2 (Pseudomonas sp.), OFT5 (Pseudomonas sp.), and Cab57 (Pseudomonas protegens), exhibited strong inhibitory activity against C. ilicicola mycelial growth. Some antifungal activity was also observed in the culture supernatants of these Pseudomonas strains. Greenhouse soil inoculation tests showed that application of OFT2, OFT5, and Cab57 cultures around soybean seeds after seed sowing significantly reduced the severity of RCR, as shown by up to 40% reduction in C. ilicicola fungal growth in the roots and 180–200% increase in shoot and root fresh weights compared to the water control. Our results suggest that OFT2, Cab57, and OFT5 produce potent antifungal compounds against C. ilicicola, thereby showing considerable potential for the biological control of C. ilicicola during soybean production.

Soybean red crown root rot (RCR), caused by the soil-borne fungal pathogen, Calonectria ilicicola, is a destructive disease observed in soybean fields worldwide. The disease is characterized by root rot, damping-off of young seedlings, and early defoliation 1,2 . It occurs mostly when seeds are grown in poorly drained soils with high clay content and/or when fields are subjected to temporary flooding and ponding. The claying nature of such soybean fields is favorable for paddy cultivation, making them prone to short-term waterlogging owing to poor soil drainage 3 . Soybean RCR is a major limiting factor responsible for low soybean grain yield in Japan, as more than 80% of soybean crops are grown in paddy-converted fields in Japan 4 . Soybean yield losses due to RCR are estimated to range from 25 to 30% 5,6 to as high as 50% depending on the environmental conditions that favor fungal colonization 2 .
Soil-borne pathogens pose a great challenge in crop production as most of them are very difficult to control via conventional agronomic practices, such as the use of resistant cultivars and synthetic fungicides and crop rotation 1,7,8 . The number of resistant plant varieties is limited, and the delivery of fungicides around the roots is inefficient, especially during the late stages of plant growth. In addition, excessive use of synthetic fungicides can result in the emergence of new pathogen isolates with fungicide resistance 9 . As with most other soil-borne pathogens 7,8 , C. ilicicola is very difficult to control due to its wide host range and longevity in soil and/or plant residues 10 . C. ilicicola infects 15 plant species 11 and can survives as microsclerotia in the soil for at least 7 years under natural conditions 12,13 . Currently, no resistant soybean cultivars or effective fungicides are available for the control of C. ilicicola 10 .
Biological control using beneficial microorganisms is receiving scientific and commercial attention as a promising alternative or a supplemental strategy for the management of soil-borne pathogens 9,14 . Many microbial strains, including Trichoderma fungi as well as Bacillus and Pseudomonas bacteria, have been used as biological control agents (BCAs) 15,16 . Most BCAs colonize the root surface (rhizosphere), whereas some can also enter the root interior and establish endophytic populations 17 17,18 . BCAs can directly suppress pathogen growth via the production of inhibitory antibiotic chemicals and competition for nutritional resources 14,[17][18][19] . Various antibiotic chemicals, including iron-chelating compounds (siderophores) and antibiotics, have been identified 14,18,19 . In addition, many BCAs have been found to produce and emit microbial volatile organic compounds (mVOCs) that are directly toxic to soil pathogens [19][20][21] . For instance, several cyanogenic Pseudomonas strains have been found to inhibit the tobacco black root rot-causing fungal agent Thielaviopsis basicola 22 and potato late blight-causing oomycete agent Phytophthora infestans 23,24 by producing antifungal mVOCs including the volatile respiratory inhibitor hydrogen cyanide (HCN). So far, more than 1300 mVOCs have been identified, with the major chemical classes being alcohols, ketones, aromatic compounds, terpenes, organic acids, esters, aldehydes, sulfur compounds, alkanes, and nitrogen compounds 19 . Among them, dimethyl disulfide has been the most extensively studied and successfully patented and commercialized as a soil fumigant (Paladin™) in greenhouses and open fields (Paladin Technical US EPA Reg. No. 55050-3) 19,20 .
In soybean, several bacterial and fungal strains have been reported for their biological control effects against Phytophthora sojae 25 , Sclerotinia sclerotiorum 26,27 , Fusarium solani 28 , Rhizoctonia solani [28][29][30] , Pythium aphanidermatum 31 , Phytophthora nicotianae 32 , and Sclerotium rolfsii 33 . Several Pseudomonas bacterial strains have been found to produce antifungal mVOCs against S. sclerotiorum, a fungal pathogen with a broad host range of over 400 plant species including soybean 26 . Gao et al. 34 reported that the application of the rhizobium strain, Bradyrhizobium sp. BXYD3, or the arbuscular mycorrhizal fungus (AMF), Glomus mosseae, from maize roots (Zea mays L.) significantly decreased the occurrence and development of RCR in soybean roots. Interestingly, the root exudates of soybean plants inoculated with Rhizobium and/or AMF significantly inhibited C. ilicicola mycelial growth, suggesting that inoculation with these microbes promotes the production of antibiotic substances in soybean plants 34 . We have previously reported the isolation of endophytic 35 and rhizosphere 36,37 bacteria belonging to diverse genera from different plant species inhabiting Japan. In this study, we evaluated the antifungal activities of these bacterial strains against the fungal pathogen, C. ilicicola, and identified three Pseudomonas bacterial strains (OFT2, OFT5, and Cab57) with strong antifungal activity, which may aid in the development of BCAs for the effective and eco-friendly management of RCR in soybean production.
The impact of bacterial VOCs on the mycelial growth of nine different C. ilicicola isolates with different pathogenic properties obtained from different prefectures in Japan 10 was tested using the double-plate chamber method by growing the bacteria and fungi in the same atmosphere, but physically separated from each other, which is the most widely used method for the in vitro assessment of VOC-mediated microbial interactions. The 13 bacterial strains showed significantly different inhibitory effects on C. ilicicola growth ( Fig. 1; Table 2). Among the 13 bacterial strains tested, two Pseudomonas sp. strains (OFT2 and OFT5) and one Pseudomonas protegens strain (Cab57) showed particularly high average inhibition rates (≥ 35%) against mycelial growth of all nine C. ilicicola isolates: 13-84% and average 46% for OFT2; 17-86% and average 35% for OFT5; and 30-86% and average 47% for Cab57. Interestingly, the growth inhibition effects of the three Pseudomonas strains were more evident against the highly virulent C. ilicicola isolates, UH2-1, AID1-12, and Y11-1b 10 , with inhibition rates of 57, 58, and 84% by OFT2, 36, 36, and 86% by OFT5, and 50, 53, and 86% by Cab57, respectively ( Fig. 1; Table 1. Bacterial strains used in this study, which were isolated from various plant species in different prefectures (localities) in Japan. www.nature.com/scientificreports/ Table 2). All remaining bacterial strains showed an average inhibition rate of ≤ 31% ( Fig. 1; Table 2). Meanwhile, a Pantoea sp. strain (HK1) and a Streptomyces sp. strain (MF7) showed high inhibition activity against the C. ilicicola isolate Y11-1b of 78 and 64%, respectively. We prepared bacterial culture supernatants of the three Pseudomonas strains (Cab57, OFT2, and OFT5) to examine whether these bacteria also produce and secrete antifungal substance(s) into the surrounding environment using the C. ilicicola isolate, UH2-1. The results showed that the culture supernatants of all three bacterial strains significantly inhibited C. ilicicola mycelial growth compared with the mock control (Fig. 2).
Evaluation of the biological control activities of the Pseudomonas strains against C. ilicicola infection. As the Pseudomonas strains, OFT2, OFT5, and Cab57, showed strong antifungal activity against C. ilicicola (Figs. 1 and 2; Table 2), we further investigated whether these bacterial strains exhibited any biological control activity in soybean plants against RCR caused by C. ilicicola (UH2-1). Compared with the disease-free mock control, a marked reduction in plant growth parameters, including plant height and fresh weight, was observed in C. ilicicola-inoculated water control plants at both 2-WPI (Figs. 3A, 4A-C) and 4-WPI (Figs. 3B, 4D-F). In contrast, seed application with OFT2, OFT5, and Cab57 significantly alleviated the negative impact of C. ilicicola infection on soybean plant growth at both sampling time points (Figs. 3A,B; 4A-C,D-F), although it could not restore the growth to the levels of the mock control. The negative impact of C. ilicicola infection was most drastic in the roots, as shown by the short height and small volume of the roots compared to the water control (Figs. 3A,B, 4C,F). Moreover, seed application of OFT2, OFT5, and Cab57 significantly reduced the root damage, as shown by the significantly recovered root volume and (Fig. 3A,B) fresh weight (Fig. 4C,F). www.nature.com/scientificreports/ Consistently, the relative fungal growth of C. ilicicola was significantly reduced by the application of the Pseudomonas strains when compared with the water treatment (Fig. 3C,D). The highest reduction was observed for OFT2 (60%), followed by Cab57 (54%) and OFT5 (34%) at 2-WPI (Fig. 3C). No significant differences in the reduction of relative fungal growth were observed between the Pseudomonas strains. A similar trend was observed at 4-WPI, where the Pseudomonas strains reduced the relative fungal growth by 42% for Cab57, 27% for OFT5, and 26% for OFT2 (Fig. 3D).
Effects of the Pseudomonas strains on plant growth. In the control plants (not inoculated with C. ilicicola), no negative effects of the Pseudomonas strains were observed at either 2 or 4 weeks after seed sowing (Fig. 4). A significant increase in the fresh weights of the roots by OFT2 and the roots and shoots by OFT5 was observed at 4 weeks after seed sowing (Fig. 4E,F). No such effect was observed with Cab57.

Discussion
Biological control is important as an eco-friendly and practical approach for plant disease management in various crops, particularly for controlling soil-borne pathogens 9,14 . Several bacterial and fungal isolates have been isolated and studied for the biological control of various soybean diseases [25][26][27][28][29][30][31][32][33] , but no BCAs with practical and  www.nature.com/scientificreports/ commercial potential for C. ilicicola control have been reported. Application of rhizobia and/or AMF alleviates RCR severity in soybean roots 38 . However, further investigation is needed on the overall RCR control effect to use these microbes as BCAs, as C. ilicicola can also invade the roots via the nodules, which may lead to even more severe RCR symptoms 39 . Moreover, it is technically challenging and expensive to in vitro propagate obligate biotrophs, including AMF, for practical use 40 . The success of this study in identifying the Pseudomonas strains (OFT2, OFT5, and Cab57) with strong biological control activity against C. ilicicola will aid in the development of effective BCAs to control soybean RCR. These findings may be particularly significant for soybean production in Japan, where RCR is one of the major limiting factors for soybean grain yield 4 . The Pseudomonas sp. OFR2 and OFT5 are endophytic bacteria isolated from carrot and turnip, respectively, and both express ACC deaminase 35 . Inoculation of OFT5 into tomato seedlings can enhance their salt stress tolerance by reducing stress-related ethylene production 41 . Pseudomonas protegens Cab57 is isolated from the rhizosphere of Shepherd's purse 37 , and exerts biocontrol activity against damping-off and root rot caused by Pythium ultimum in cucumber plants 36 . In this study, three Pseudomonas strains produced and emitted mVOCs with potent antifungal activity against nine different C. ilicicola isolates ( Fig. 1; Table 2). Each bacterial strain exhibited different growth inhibitory effects on different C. ilicicola isolates, and some bacterial strains, HK1 and MF7, showed a high inhibition rate for a particular C. ilicicola isolate (Y11-1b) ( Table 2). In addition, the three selected Pseudomonas strains (OFT2, OFT5, and Cab57) had high growth inhibitory effects against C. ilicicola isolates with high virulence (UH2-1, AID1-12, and Y11-1b) in soybean plants 10 (Fig. 1; Table 2). Whether this biased inhibitory effect on different C. ilicicola isolates has any biological significance remains to be clarified. Taken together, these results indicate some genetic and biochemical variations among the Pseudomonas and C. ilicicola isolates, which determine the outcomes of the interactions between individual bacterial and fungal isolates. The Pseudomonas strains (OFT2, OFT5, and Cab57) were also shown to produce and secrete antifungal substance(s) against C. ilicicola (UH2-1) (Fig. 2), indicating that these bacteria can suppress C. ilicicola growth by producing antifungal mVOCs and secretory metabolites. In support of this notion, genomic analysis of strain Cab57 revealed that it harbors the gene clusters for production of HCN 37 , a potent antifungal mVOC [22][23][24] , and the www.nature.com/scientificreports/ antibiotics 2,4-diacetylphloroglucinol, pyrrolnitrin and pyoluteorin etc. 16,37 . It would be important and interesting to assess the role of these chemical compounds in the inhibition activity on C. ilicicola growth. In soil inoculation experiments, C. ilicicola inoculation caused severe root rot and growth retardation in the soybean seedlings. In contrast, co-inoculation of C. ilicicola with a Pseudomonas strains (OFT2, OFT5, or Cab57) significantly reduced C. ilicicola proliferation in the roots (Fig. 3C,D) and rescued the plant growth inhibition caused by C. ilicicola infection to some extent (Fig. 3A,B; Table 2). These results demonstrate that the Pseudomonas strains, OFT2, OFT5, and Cab57, have strong biocontrol activities against C. ilicicola, which may be used for the development of BCAs to manage RCR during soybean production. Our results suggest that the mechanism of biocontrol activity of these bacteria is at least partly associated with the antagonistic suppression of C. ilicicola growth via the production and release of antifungal mVOCs ( Fig. 1; Table 2) and secretory metabolites (Fig. 2). Whether these bacterial strains are also capable of inducing host resistance remains to be determined in future studies.
No negative effects of the Pseudomonas strains (OFT2, OFT5, and Cab57) on soybean plant growth were observed (Figs. 3A,B, 4), which is important for their practical use as BCAs in RCR management. Rather, an increase in the fresh weight of the roots by OFT2 and the roots and shoots by OFT5 was observed (Fig. 4E,F). These growth-promoting effects of OFT2 and OFT5 may be attributed to their ACC deaminase activity, as www.nature.com/scientificreports/ ethylene generally reduces the plant growth 35,41 . On the other hand, the strain Cab57 showed no significant plant growth promoting effect, even though it also contains a homologue of ACC deaminase gene in its genome (PPC_RS20245) 37 . Therefore, further study remains to clarify the contribution of ACC deaminase activity to soybean plant growth.
In summary, we found that three Pseudomonas strains, OFT2, OFT5, and Cab57, significantly inhibited the development of soybean RCR caused by the fungal pathogen, C. ilicicola. This biological control effect relies on the antagonistic suppression of C. ilicicola growth via the production and release of antifungal substances. These bacterial strains may provide a basis for the development of BCAs for the effective management of soybean RCR. However, the specific substance(s) responsible for the suppression of C. ilicicola growth and the efficacy of these bacterial strains in controlling RCR in actual soybean fields require further elucidation in future studies.

Methods
Plant material and growth conditions. Soyabean (Glycine max) cv. Enrei was used for all experiments in this study. Enrei is a C. ilicicola-susceptible cultivar popularly cultivated in Hokuriku and Northeast regions of Japan. The seeds of Enrei were obtained from the Institute of Agrobiological Sciences, NARO, Japan. All the experimental procedures including the collection of plant material complied with institutional, national and international guidelines and legislations.
The seeds were pre-conditioned in a moisture-saturated plastic box for 24-48 h at 25 °C. The seeds were then sown in commercially available pre-fertilized and granulated soil (Nippi No.1, Nippon Hiryo, Tokyo, Japan) in 144-cm 2 plastic pots at a depth of 20 cm. Five seeds were sown per pot (12 cm × 12 cm × 20 cm; 1500 mL) and grown in a greenhouse at 25 °C and 50% relative humidity. All soils used in this study were autoclaved at 120 °C for 1 h one day before seed sowing to eliminate any effects of other soil pathogens.
C. ilicicola culture and inoculation. Fungal mycelia of nine C. ilicicola isolates ( Table 2) were cultured on potato dextrose agar (PDA) plates at 25 °C for 1-2 weeks or until fungal mycelial growth reached the edges of the Petri plates (9 cm) 2,10 .
The C. ilicicola isolate, UH2-1, was used for the inoculation of soybean (Enrei) as described previously 10,42 . Briefly, 5-8 pieces (~ 5-mm cubes) of PDA with vigorously growing C. ilicicola mycelia were placed in a 500-mL flask containing 200 g of wheat bran-vermiculite medium (wheat bran/vermiculite/water 1:1:3, w/w/v) and incubated at 26 °C for 10-14 days, until the fungal mycelia fully covered the medium 42 . This culture was used as the inoculum, and an inoculum-soil mixture was prepared by mixing the inoculum with Nippi No.1 soil to generate a concentration of 1% (w/v). The soil mixture was then filled in plastic pots (12 × 12 × 20 cm; 1500 mL), into which five seeds were sown per pot.
Culture of bacterial strains. The bacteria (Table 1) were cultured overnight on tryptic soy agar (TSA) plates at 28 °C. For biological control assays, bacteria were cultured in tryptic soy broth (TSB) medium with shaking (150 rpm) at 28 °C for 24 h.

Measurement of antifungal effects of bacterial VOCs.
The possible antifungal effects of mVOCs produced by the bacterial strains against C. ilicicola were examined using the double-plate chamber method 43,44 . Nine C. ilicicola isolates were used in this study (Table 2). Briefly, 10 μL of overnight bacterial culture in TSB broth (OD 600 = 0.4) was spread on the surface of a TSB agar plate (5.2 cm in diameter) and incubated overnight at 25 °C. On the other hand, a 5-mm diameter mycelial plug of C. ilicicola was inoculated individually at the center of the PDA agar plate (5.2 cm in diameter). The TBS broth agar plate with a bacterial strain was placed onto the PDA agar plate inoculated with a C. ilicicola isolate so that the two plates faced each other. The contact surfaces of the two plates were sealed with parafilm to obtain a double-plate chamber, and the plates were incubated at 25 °C in the dark for 10-14 days. The average distance between the surfaces of the two plates was 1.5 cm. The fungal growth rate was represented as colony diameter (cm). A double-plate chamber without bacterial strains was used as the control. The experiment was repeated thrice, with four replicates each. The percentage inhibition of fungal growth was calculated as follows: where C represents the colony diameter (mm) in the mock plate (with water as a control) and T represents the colony diameter (mm) in the bacterial plate.
Measurement of antifungal effects of bacterial culture supernatant. The bacterial culture supernatants of the three Pseudomonas strains (Cab57, OFT2, and OFT5) were investigated for their antifungal activities against C. ilicicola. Bacterial culture supernatants were prepared according to the method described by Pethani 45 , with slight modifications. The Pseudomonas strains (Cab57, OFT2, and OFT5) were cultured on TSA plates at 28 °C for 24 h. The TSA medium containing the bacterial culture was homogenized by passing through a syringe several times and mixed with an equal volume of sterilized water. The slurry mixture was centrifuged at 10,000×g for 60 min, and the supernatant was filtered through a 0.22-μm Millipore filter (Whatman ® 9911-1302 Syringe filter) to remove any remaining bacteria. A filter paper (Whatman) was soaked in 4 mL of bacterial supernatant and placed in a petri plate (9-cm diameter). A small agar plug of C. ilicicola culture was inoculated onto the filter paper at the center of the plate and incubated at 28 °C for 7 days. The supernatant prepared from the TSA medium without bacterial culture was used as the control (mock).